Derlin-1 facilitates the retro-translocation of cholera toxin
Derlin-1 facilitates the retro-translocation of cholera toxin
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DOI：
10.1091/mbc.e07-08-0755
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发表时间：
2008-03-01
影响因子：
3.3
通讯作者：
Tsai, Billy
中科院分区：
文献类型：
--
作者：
Bernardi, Kaleena M.;Forster, Michele L.;Tsai, Billy
霍乱毒素（CT）通过其受体结合B亚单位（CT B）从质膜运输到内质网（ER）而使细胞中毒。在该隔室中，催化性A1亚基（CTA 1）通过蛋白质二硫键异构酶（PDI）展开，并逆向易位至胞质溶胶，在胞质溶胶中触发信号级联反应，导致分泌性腹泻。CT如何靶向ER膜中的反向易位位点以启动易位尚不清楚。使用半透性细胞逆易位试验，我们证明，显性负Derlin-1-YFP融合蛋白衰减ER到CTA 1的胞质溶胶运输。Derlin-1与CTB和ER伴侣PDI相互作用，如通过免疫共沉淀实验所评估。体外膜结合试验表明CTB刺激未折叠的CTA 1链与ER膜结合。此外，完整细胞与CTB的中毒稳定了Derlin-1依赖性底物的降解，表明CT使用Derlin-1途径。这些发现表明，Derlin-1促进CT的反向移位。CTB可能通过将全毒素靶向至Derlin-1，使Derlin-1结合的PDI解折叠A1亚基并使其准备运输而在此过程中发挥作用。
Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.